Filter-aided N-glycan separation (FANGS): a convenient sample preparation method for mass spectrometric N-glycan profiling

Abstract

We have developed a simple method for the release and isolation of glycoprotein N-glycans from whole-cell lysates using less than a million cells, for subsequent implementation with mass spectrometric analysis. Cellular protein extracts prepared using SDS solubilization were sequentially treated in a membrane filter device to ultimately release glycans enzymatically using PNGase F in the volatile buffer ammonium bicarbonate. The released glycans are recovered in the filtrate following centrifugation and typically permethylated prior to mass spectrometric analysis. We call our method "filter-aided N-glycan separation" and have successfully applied it to investigate N-glycan profiles of wild-type and mutant Chinese hamster ovary cells. This method is readily multiplexed and, because of the small numbers of cells needed, is compatible with the analysis of replicate samples to assess the true nature of glycan variability in tissue culture samples.

Figure 1

Schematic of FANGS protocol for cell lysate preparation from cultured cells, N-glycan release, and sample handling in membrane filter devices. (a) Extraction of glycoproteins from whole cells and (b) release and permethylation of N-glycans.

Figure 2

(a–c) Positive-mode MALDI mass spectra of permethylated FANGS-released N-glycans from 2.5 μg fetuin: (a) 1/10th of the N-glycan sample loaded onto the MALDI plate for analysis by FT-ICR and (b,c) 1/20th of the N-glycan sample loaded onto the MALDI plate for analysis by TOF-MS. (d–f) MALDI-TOF mass spectra of permethylated FANGS-released N-glycans from (d) WT HeLa cells, (e) HeLa cells treated with 10 μg/mL swainsonine, and (f) COG4KD cells. Each spectrum is derived from cells collected off one confluent 10 cm dish, approximately 2 to 3 × 10⁶ cells. Peaks derived from contaminating cellulose oligomers are indicated with *. N-Glycan structures are denoted following the conventional symbols described in ref (36). Peak intensities in each spectrum are normalized to the most intense signal in the spectrum.

Figure 3

(a,b) MALDI-TOF mass spectra of permethylated FANGS-released N-glycans from (a) WT CHO and (b) ldlB cells. Cellulose oligomer contamination peaks are indicated with *. (c) Bar chart representing the relative intensities of the MALDI-TOF-MS signals for the different N-glycan species detected in WT, ldlB, and ldlC cells. The glycan intensities in each individual spectrum are normalized to the sum of intensities in a given spectrum for the subset of glycans that are common to all three cell lines. Standard errors of the mean are shown for WT CHO cells (n = 7), ldlB cells (n = 5), and ldlC cells (n = 4). Statistically significant differences between relative glycan peak intensities are indicated: *** for p < 0.001, ** for p < 0.01, and * for p < 0.05. None of ldlB- and ldlC-derived glycan samples showed a significant difference when compared with each other. The differences between WT and the two mutants showed similar significance, except for the Hex₃HexNAc₄ and the Fuc₁Hex₄HexNAc₄ species. For both of these species, the ldlC-derived glycans showed differences of lower significance when compared with WT than for the ldlB compared with WT. For the Fuc₁Hex₄HexNAc₄ species, the difference between WT and ldlC was not significant.

Figure 4

MALDI-TOF mass spectrum of permethylated FANGS-released N-glycans from 3.8 × 10⁵ WT CHO cells (one well of a six-well plate). Peak intensities are normalized to the most intense signal in the spectrum.